Aspartate is the most highly consumed amino acid by the mouse blastocyst and is involved in the regulation of carbohydrate metabolism by acting as the rate-limiting factor of the malate-aspartate shuttle. Furthermore, data on global amino acid profiling reveal that aspartate consumption by blastocysts is significantly higher in groups of kinetically faster embryos. The aim of this study was to determine how aspartate consumption affects carbohydrate metabolism. As metabolism of these substrates has been shown to be different in kinetically different embryos, the mRNA levels of genes involved in aspartate and carbohydrate metabolism were also determined.
We developed an ultramicrofluorescence assay to measure aspartate uptake by individual mouse blastocysts over a range of aspartate concentrations. In vivo day 4 blastocysts were flushed from CBAxC57BL/6 mice and incubated in drops of medium over increasing aspartate concentrations (0mM, 0.1mM, 1.0mM, 10.0mM). Aspartate consumption increased with respect to concentration (P<0.001). Glucose uptake, standardized to blastocyst cell number increased in proportion to aspartate concentration (P<0.01), while lactate production displayed a similar trend (P<0.08). These alterations in glucose and lactate levels did not affect the overall glycolytic rate (percentage of glucose forming lactate).
In vitro fertilized CBAxC57BL/6 zygotes were cultured individually in a time-lapse incubator. Blastocysts were divided into quartiles based on 2-cell cleavage time and gene expression assessed by real-time PCR. Results showed that Slc2a1, PKM and GOT1 mRNAs were expressed at significantly higher levels in kinetically slower blastocysts. This suggests a compensatory response in metabolic regulation, given our previous data showing that slower, less viable, blastocysts consume less glucose and aspartate (Lee et al., 2015 Hum Reprod 30: 543-552).
These findings demonstrate that aspartate is involved in the regulation of glucose metabolism and embryo kinetics. Aspartate uptake may therefore be used as a potential biomarker for embryo selection to increase IVF success outcomes.